Week 8

CONGRATS WE ARE FINISHED

MONDAY – SEMESTER PROJECT DUE + PURE CULTURE

TUESDAY – LAB PRACTICAL 2 (14-26)

WEDNESDAY –

THURSDAY – FINAL LECTURE EXAM

 

Exercise 14:  Use of Selective & Differential Media & Replica Plating Techniques
1. Define these terms:
a. Complex media – substance containing the nutrients required for microbial growth, but in crude form, meaning their exact quantities are unknown (eg. Nutrient Agar & Blood Heart Agar)
b. Defined media  – substance containing required nutrients for microbial growth, in relatively pure chemical form & exact types and amounts known.
c. Enriched media (also list examples) – Media containing cells, tissue or tissue fractions (Blood Agar & Chocolate Agar)
d. Differential media – media supporting the growth of various types of organisms while distinguishing between different forms of organisms (MAC, MSA, T-7)
e. Selective media – media that supports the growth of specific organisms and inhibiting the growth of others. (MAC, T-7)
2. Explain the differential and selective characteristics of each media shown below.  Know which
pH indicator is used in each media and how to interpret the results (see table called “Selective
& Differential Media”):
a. Mannitol Salt Agar (MSA) – Selective media for isolation of pathogenic & non-pathogenic Staphylococci. (Many other organisms are inhibited by the 7.5% salt content). It is also a differential on basis of mannitol fermentation. pH indicator =phenol red.

Yellow = acidicPink = neutral →  Purple = alkaline
b. MacConkey Agar (MAC) – Selective medium (based on chemicals) for Gram-Negative organisms, inhibiting growth of Gram-Positive. Differential as well for those that can ferment lactose from those that cannot. pH indicator = neutral red. (+) organisms will accumulate red in or around their colonies. (ferment lactose)
c. Eosin Methylene Blue Agar (EMB) – Selective Media (based on chemicals) for growth of Gram-negative bacilli. Differentiates between lactose fermenting colonies and non-lactose fermenting. Lactose fermenting colonies will be a dark/irridescent green color. (+)
d. Tergitol 7 Agar (T-7) – Selective media (based on chemicals) for E. coli & other members of coliform group. Inhibits gram-negative spore-formers and Gram-positive organisms. pH indicator = Bromothymol blue

(lactose fermenter) Yellow = acidicGreen = neutral →  Blue = alkaline (non-lactose fermenter)
3. Explain the function (e.g. benefits) of replica plating. Technique allowing for rapid, efficient transfer of multiple colonies all at one time.
4. What type of material (cloth) was used for obtaining bacteria from the master plate? Why is
this material useful for obtaining bacteria? Velvet; the tiny hair-like projections of velvet readily pick up bacterial cells.

Exercise 15-A, B, & C:  Physiological Tests
1. Know the following information about each of the physiological tests (see tables called “Tests
for Gram-positive Organisms” and “Tests for Gram-negative Organisms”):
a. Purpose (question asked with test)
b. Key ingredients, including pH indicator
c. Possible outcomes (results)
d. How to interpret each possible outcome (conclusions)

Oxidase Test – Q:Does organism have Cytochrome C? Positive; No color change is negative I: Oxidase Reagent
Catalase Activity TestQ: Does organism have catalase? Bubbles (+), No Bubbles (-) I: 3% Hydrogen Peroxide
O/F Test: Q: Can organism ferment glucose to acid & gas? Yellow (+) Green (-)  I: Glucose & pH indicator: Bromethymol Blue
Simmon’s Citrate  Q: Can organism use citrate as sole carbon source? Blue (+) Green (-) I: Sodium Citrate & pH indicator: Bromythymol Blue
Urea Agar Test Q: Does organism have urease? Pink (+) Peach (-) I: Urea & pH indicator: phenol red
MR-VP Test Q: MR – Does organism perform mixed acid fermentation? Red (+) Brown (-) I: glucose, buffer & pH indicator: methyl red Q: VP – Does organism perform butanediol fermentation? Red (+) Brown (-) I: Glucose, Barritt’s reagents A & B
Lysine Test Q: Does organism decarboxylate lysine and formate amine? Control tube must turn Yellow for test to work. Purple (+) Yellow (-) I: Glucose, Lysine (not in control tube) and pH: bromcresol purple
Coagulase Test Q: Does organism have coagulase? Thickens (+) Liquid (-)
Esculin Test Q: Can organism hydrolize esculin? Black (+) Brown (-)

(TSI) Triple Sugar Iron Test

  • Q: Does organism ferment more than 1 sugar?
  • Q: Does the organism produce H2S (Hydrogen Sulfide)?
  • Ingredients: Glucose, Sucrose, Lactose
  • Ingredients: Iron
  • (+): Red slant/yellow butt = organism ferments glucose only; or yellow slant/yellow butt = organism ferments more than 1 sugar (crack = gas fermentation)
  • (+):black agar= organism produces H2S
  • (-): red slant/red butt = does not ferment any sugar
  • (-): no black = no H2S production.

(SIM) Sulfur Indole Motility Test

  • Q: Is organism motile?
  • Q: Does organism produce indole from catabolism of tryptophan?
  • Q: Does organism produce H2S?
  • Ingredient: Low concentration of agar
  • Indgredient: Tryptophan + Kovac’s Reagent (day 2)
  • Ingredient: Iron, forming iron sulfide precipitant
  • (+): “cloudiness” away from stab = motile
  • (+): Kovac’s forms cherry red color =  organism produces indole during catabolism of tryptophan.
  • (+): Black = organism produces H2S
  • (-) results are opposite of positive, (no cloudiness, nor bright red, not black)
  • Procedure: Using NEEDLE, make straight stab to bottom of Agar tube.

Carbohydrate Utilization Test

  • Q: Does this organism catabolize _______________?
  • Ingredients: Select Carbohydrate
  • pH indicator: phenol red
  • (+): Turns media yellow
  • (-): Stays red/pink
  • Procedure: use loop & stab to bottom of tube.

2. What are enteric bacteria?  Be able to recognize these enteric bacteria on the list of organisms
for our physiological unknown #1 samples (see table):
Citrobacter fruendii  Enterobacter aerongenes
Enterobacter cloacae  Escherichia coli
Klebsiella oxytoca   Klebsiella pneumonia
Proteus mirabilis   Proteus vulgaris
Salmonella choleraesuis  Serratia marcescens
Serratia odorifera

Note that these enterics are Gram-negative rods and also O-F positive (fermentative)

3. Be able to recognize the Gram-positive bacteria on the list of organisms for our physiological
unknown #1 (see table):
Bacillus cereus   Bacillus sphaericus
Bacillus subtilis   Enterococcus faecalis
Staphylococcus aureus  Staphylococcus epidermidis
Streptococcus mitis  Streptococcus pyogenes

Exercise 16:  Application of the Polymerase Chain Reaction (PCR) in Bacterial Identification
1. List two applications of PCR. Make many copies of particular gene or DNA segment.
2. PCR is in an in vitro version of what process? DNA Replication
3. List the ingredients (reagents) that are necessary for conducting a PCR (see bottom of p. 197)

Template DNA, 2 Primers, 4 types of Nucleoside triphosphates, Taq Polymerase, Buffer, Distilled water.
4. A special type of DNA polymerase is used for PCR.  What type of DNA polymerase is this and
why is it used?

Taq polymerase is used. It contains Thermus aquaticus which is a heat-stable organism found in the hot spring of Yellowstone National Park. This one is specifically used because since it is heat stable it will not be killed at high heats during the denaturation processes in the Thermocycler.
5. Know the names of the primers we used for PCR.

8 Forward, 1494 Reverse.
6. Which gene was these primers targeting/amplifying?  How long is this gene?  Why is this gene
commonly used for PCR?

16s Ribosomal RNA; 1484 nucleotides long.
7. What was the source of the template DNA for our PCR?

DNA from PUK 2
8. Nucleoside triphosphates (dNTPs) were used for PCR.  What is the function of the multiple
phosphates on the dNTPs?

They provide the energy for the DNA synthesis when the phosphate bonds break.
9. Note about nucleosides vs. nucleotides:
a. Nucleoside = nitrogenous base plus sugar
b. Nucleotide = nitrogenous base plus sugar plus phosphate(s), also called nucleoside
phosphate(s) 
10. What was the purpose of heating (boiling) the unknown samples before performing PCR (see
protocol on p. 197)? Releases DNA from cells and also denatures proteases and nucleases that could interfere with the PCR.
11. What was the purpose of vortexing Gram-positive unknown samples with glass beads before
performing PCR (see protocol on p. 197)? Because gram-positive organisms have a thick peptidoglycan wall, vortexing with beads will help break the wall allowing the DNA to release from the cells.
12. What is a thermocycler?   Device that is programmed to alternately raise and lower the temperature to facilitate the PCR reaction.
13. Know the following information about the thermocycler settings that we used for PCR:
a. How many cycles are performed by the thermocycler?  How long is each cycle? 35 Cycles.
b. Know what happens during each stage of a cycle and the temperature at which each
stage occurs (denaturation stage, annealing stage, and extension stage).

1. Denaturation, 4 min @ 94°C – Separates cell DNA strands.

2. Annealing, 45 sec @ 55°C – allows the primers to attach to the template DNA near beginning and end of 16s rRNA gene.

3. Extension, 2 min @ 72°C – this is when the nucleotides get attached (bonded) to old strand, forming new DNA molecules

4. Denaturation, 30 sec @ 34°C – separates cell DNA strands.

Exercise 17:  Automated Nucleotide Sequencing and Electropherogram Evaluation
1. Define nucleotide sequencing. determining the sequence or arrangement of DNA nucleotides (bases) in a sample of DNA molecules.
2. A small amount of dideoxyribonucleoside triphosphates (ddNTPs) are included in nucleotide
sequencing reactions.  Explain the function of ddNTPs. because they lack a hydroxyl group (OH-) on the 3rd carbon of its sugar, when used during PCR they are unable to form phosphodiester bonds therefore terminating DNA synthesis.
3. Answer the following questions about electropherograms:
a. Define electropherogram. Data collection software and computer analysis used to generate a visual record of a DNA sequence.
b. What program was used to generate the electropherograms for our physiological
unknowns? For Peaks, Four Peaks.
c. What does each peak on an electropherogram represent? A specific base (nucleotide)
d. How many electropherograms did we generate for each unknown sample?  What was
the source of each electropherogram? 3 electropherograms from UC Davis DNA Sequencing Facility
e. Why was it necessary to flip the 1492-Reverse sequence before copying it to the word
file? Because it recorded the sequence backwards, so in order for it to be matched with the 8-forward in the right order we had to flip it.
4. Know the general procedure that we performed during Exercise 17 (see pp. 200-201).
5. During this exercise, we were trying to make a word file containing a contiguous sequence of
nucleotides.  Define contiguous sequence. Sequence of the nucleotides obtained from the electropherogram, in which we have connected the sequences together, editing out the less defined peaks. 

Exercise 18: Genomics, Proteomics & Bioinformatics
1. Know the general procedure that was used for identifying your physiological unknown #2
sample (see Section A at the bottom of p. 204).
2. BLAST is a program/service offered by the National Center for Biotechnology Information.
What does BLAST stand for?  Explain what happens when you run BLAST on the nucleotide
sequence for your unknown. Basic Local Allignment Search Tool. Compares your nucleotide or amino acid sequencing to others in public databases to identify your organism.
3. Be able to interpret BLAST results on the NCBI web site, as you did for your physiological
unknown #2 sample.  Remember there’s a BLAST tutorial on the Geospiza site
(http://www.geospiza.com/education/).  On the education page, click on animated tutorials and
then click on BLAST for beginners.
Exercise 19: The Miniscreen- Rapid Isolation of Plasmid DNA
1. Define the following terms:
a. Cloning vector – Segment of DNA (usually a plasmid) that can be used to make many copies of a gene of interest inside living cells (usually bacteria)
b. Copy number (plasmids) – Total # of any one type of plasmid within a cell
c. Expression vector – Segment of DNA that can be used to make many copies of a gene of interest inside living cells but can also express the gene (make product)
d. Marker gene – Gene that makes a product that can be used to identify cells that contain the plasmid, typically nutritional genes or antibiotic resistance factors.
e. Plasmid – loops of extrachromosomal CCC DNA and replicate independently
2. During Exercise 19, each student received an E. coli sample containing one of three types of
plasmids.  List the three types of plasmids that we were trying to isolate during Exercise 19
and know their general characteristics (approximate size; genes; coning vs. expression
vector).

E. coli strain JM83 with plasmid pUC19: Cloning Vector. 2686 bp long. Marker gene – bla gene: beta lactamase, enzyme that breaks down or destroys beta lactam – antiobiotics such as penicillin & ampicillin.

E. coli strain DH5α with plasmid pGLO:  Cloning & Expression Vector. 5400 bp long. Marker gene: GFP – Green Flourescent Protein that glows green under UV light. 

E. coli strain DH5α with pGEM: Cloning & Expression Vector. Same as pGLO w/o the GFP.
3. Know the general procedure we used to isolate the plasmids (pp. 213-214), including the
following information:
a. The Lytic mix contained NaCl and SDS.  What was the function of these reagents? They lyce the cells and SDS also lyces cells and coats proteins.
b. What was the function of the sodium acetate buffer? for DNA stabilization
c. After the first centrifugation step (step #8, p. 213), which portion of the centrifuged
material did we save- the supernatant or the pellet?  What was in the discarded
portion? supernatant saved, and pellet containing DNA discarded.
d. What was the function of the RNAase stock solution?

Exercise 20:  Gel Electrophoresis of DNA Samples
1. Explain the general purpose of gel electrophoresis. procedure that allows DNA fragments to be separated based on size and observed with the naked eye.
2. Define the terms anode and cathode. Anode = positive pole; cathode = negative pole.
3. When the current is applied to the gel electrophoresis box, DNA moves toward the
_____________________ (anode or cathode).  Explain why. Anode, because DNA has a slight negative charge so it is attracted to the positive pole.
4. Know the function of each reagent used for gel electrophoresis:
a. Agarose – purified agar – the jelly agar used to place the DNA in.
b. Bromphenol-blue (loading dye or tracking dye) – migrates at rate of 100-300 bp fragments, helps keep track of the DNA
c. DNA (PCR product DNA or plasmid DNA)
d. Ethidium bromide + xylene cyanol + glycerol (loading dye or tracking dye).
e. Lambda standard DNA precut with known restriction enzyme (e.g. HindIII)
f. TBE electrophoresis buffer
5. Know how to interpret the results of gel electrophoresis with regard to the movement/positions
of DNA fragments based on their relative sizes.

Exercise 21: Restriction Endonuclease Digestion of DNA and Restriction Fragment Length
Polymorphism
1. What are restriction endonucleases (restriction enzymes) and how are they used in DNA
Technology? Enzymes that cut DNA within a strand by breaking the phosphodiester bonds.
2. Explain how restriction enzymes are named. Names correspond to bacteria in which restrictions enzymes are found…. 1st letter: Genus name of bacteria 2nd & 3rd letter: specific epithet of bacteria 4th letter: bacterial strain.  Roman numeral: order in which it was discovered.
3. Define palindrome.
4. Define restriction fragment length polymorphism (RFLP). banding pattern that is an accumulation of DNA fragments that are the same length.
5. Which restriction enzyme was used to cut our DNA samples during Exercise 21? AluI
6. Be able to perform the computer procedure we conducted during this lab (see pp. 225-226),
including the diagram (step #9, p. 226).

! ! Summer 20
3
Exercise 22-A:  Calcium Chloride Procedure for Making Competent Cells
Exercise 22-B:  Transformation of E. coli with Plasmid DNA
1. Define the following terms related to this lab:
a. Cloning vector – segment of DNA (usually plasmid) that can be used to make many copies of a gene of interest inside living cells.
b. Competent – cells with better ability to take in DNA
c. Copy number –
d. Expression vector
e. Marker gene – gene that code for resistance to the antibiotic ampicillin and allow for detection of transformed cells on media containing this antibiotic.
f. Plasmid – Loops of extrachromasomal CCC DNA found in bacterial cells
g. Transformation – uptake of DNA from the environment
Exercise 22 A/B cont’d
2. We used two different strains of E. coli for Exercise 22-B (strain JM83 and strain DH5α).
These strains had been grown until they were in the mid-log phase of their growth curve.  Why
did we use bacteria cells in the mid-log phase? Due to their elongated shape during the mid-log phase there is more surface area in which DNA can enter.
3. What is the function of ice cold CaCl2 during the exercise (why ice cold and what does CaCl2
do)? The cold CaCl2 stabilizes the fluid cell membrane and coats it in cations attracting the negatively charged DNA to its surface for more likely uptake.
4. What is the typical transformation efficiency after treatment? 1/10 of all visible cells present become competent.
5. We used three different plasmids for Exercise 22-A.  All three plasmids have an origin of
replication and relatively high copy numbers, so all three can be used as cloning vectors.
Know the following information about each plasmid:
a. pUC19- size is 2686 bp; carries bla marker gene for “-lactamase (enzyme that breaks
down “-lactam drugs such as ampicillin)
b. pGEM- size is approx. 3500 bp; expression vector (as well as cloning vector); carries
bla marker gene
c. pGLO- size is 5400 bp; carries green fluorescent protein (GFP) gene in an inducible
operon (arabinose is inducer); carries bla marker gene
6. Know the expected results and explanation (conclusions) for each plate we streaked during
Exercise 22-B:

Exercise 23-A:  Viruses & Phage Typing
1. Define the following terms:
a. Coliphage – phages that infect E. Coli
b. Cytolytic – cell lycing bacteriophages
c. Bacteriophage – meaning “bacteria-eater” – viruses that infect bacteria
d. Phage typing – using a known bacteriophage to identify an unknown bacteria
e. Plaque – Clear “window” on a plate with a lawn where the the bacterial cells have been lyced.
2. Which coliphages were used for this exercise? Ø Coliphage x174 & Ø T2
3. Know the approximate plaque size produced by each coliphage – Ø Coliphage x174 – larger plaques (3-4mm in diameter) & Ø T2 – smaller plaques (pinpoint)
4. Which strains of E. coli were used for this exercise?  Which strain was the optimum host for
the T2 coliphage?  Which strain was the optimum host for the X174 coliphage?  If one of the
coliphages infected both strains of E. coli, did the plaques look the same in each strain?
Explain
5. Know the expected results and explanations (conclusions) for each plate we streaked during
Exercise 23A

Exercise 23-B:  Isolation and Purification of Coliphages from the Environment
Note:  The first part of this lab was completed by an instructor.  E. coli strain C were added to different
dilutions of warmed filtered sewage (simulated) and then grown on streak plates.  Sewage contains
various types of coliphages (because there’s a lot of E. coli in sewage!).  Once E. coli strain C has
been added to the sewage and grown on a streak plate, the different types of coliphages can be
distinguished from one another based on their plaque size and plaque appearance
1. Why was this particular strain (strain C) of E. coli used for this exercise? Most susceptible to viruses; and the optimum host for Ø X174, but can also be infected by Ø T2
2. Is it possible to isolate one type of coliphage from one of these streak plates?  If so, how? – yes, stab the plaque
3. Be able to calculate the individual dilution factor and the total dilution factor for each tube
shown in Fig. 23B.1
4. Define plaque-forming unit (PFU)
5. Be able to calculate the PFU per ml for a sample streak plate (including the number of stabs in
the original plaque)

Exercise 23-C:  Bacteriophage Reproduction and Plaque Formation
1. Define the following terms:
a. Adsorption
b. Vegetative phage
c. Latent period/ burst time
d. Burst size/ burst number
2. Be able to calculate PFU data (from plaque plates), plot the data on log graph paper and
calculate the burst time and burst size

Exercise 24A:  Microbial Control Methods
1. During one of the exercises pond water was boiled for 1 minute (Pasteurized) or 5 minutes
(sanitized) and then spread onto streak plates.  Explain the results of this experiment
2. What light wavelength (range) is most lethal to bacterial cells
3. Explain how the ultra violet (u-v) light experiment was performed
4. Know the expected results and explanation (conclusions) for the bacteria plates exposed to u-
v radiation
5. Define the following terms:
a. Antiseptic
b. Chemotherapeutic agent
c. Disinfectant
6. Explain how the antiseptics/disinfectants experiment was performed using filter paper disks
and be able to measure the zone of inhibition for each agent tested during this experiment

Exercise 24B:  Antimicrobial Sensitivity Testing
1. Explain the Agar Diffusion Method (Kirby-Bauer Test) for testing chemotherapeutic agents
2. Be able to measure the zone of inhibition for each agent tested during the Kirby-Bauer Test
and determine whether the bacteria is sensitive to each agent (using the table on p. 269 of the
lab syllabus)
3. Do all bacteria have the same sensitivity to a particular chemotherapeutic agent?

Exercise 26:  Composition of Blood & White Cell Study
1. List the three types of cells found in human blood.  Which of these types of cells do not have
nuclei?
2. List the five types of leukocytes that are normally observed in human blood samples and their
relative percentages.  Be able to identify each type of leukocyte in a prepared slide of human
blood

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